Serum and Plasma miRNA Analysis by qPCR

From sample to answer, learn about methods for optimal detection of miRNAs from serum and plasma

miRNA detection and quantification method

Although a variety of qPCR-based analysis solutions are currently available to quantify miRNAs, they may lack either sensitivity or specificity, have cumbersome workflows, or have limited instrument and software compatibility. To address these challenges, we have developed an miRNA analysis workflow that includes both a universal RT step and the superior sensitivity and specificity of TaqMan probe-based chemistry.

miRNAs can have extensive sequence homology and are grouped in highly conserved families of nearly identical sequence. Cross-reactivity of TaqMan Advanced miRNA Assays was tested between four hsa-let-7 miRNAs (Table 1).

TaqMan Advanced miRNA Assays exhibited zero cross-reactivity for the majority of target combinations. For hsa-let-7c that differs from hsalet-7a by a single base (G/A), results show less than 2% reactivity. Thus, TaqMan Advanced miRNA Assays are capable of discriminating closely related miRNA sequences, differing by as little as a single nucleotide.

Target

Assay

hsa-let-7a-5p

hsa-let-7b-5p

hsa-let-7c-5p

hsa-let-7d-5p

hsa-let-7a-5p

100

0

0

0

hsa-let-7b-5p

0

100

0

0

hsa-let-7c-5p

1.5

0.1

100

0

hsa-let-7d-5p

0

0

0

100

Table 1. Percent reactivity of TaqMan Advanced miRNA Assays tested against each hsa-let-7 family member and calculated based on exact-match results.

The TaqMan Advanced miRNA Assay workflow incorporates a unique miR-Amp universal amplification step, combined with proven TaqMan Assay design and chemistry, to enable consistent and reliable results down to as little as 1 pg of RNA. This level of sensitivity is required for challenging investigations such as the study of noninvasive biomarkers where circulating miRNA levels are limited.

To assess limits of detection, Invitrogen Human Liver Total RNA was diluted and 10 ng, 1 ng, 100 pg, 10 pg, and 1 pg were tested with four assays chosen to represent a range of expression levels in liver (Figure 1). TaqMan Advanced miRNA Assays and Exiqon miRCURY™ LNA™ microRNA qPCR assays were compared in parallel. The experiments were carried out according to manufacturer’s recommended protocol including amount of total RNA input.

TaqMan Advanced miRNA Assays show greater accuracy in measuring small fold-changes in serum

To determine accuracy of expression, serum samples 1 and 2 were generated by spiking three different synthetic miRNA targets (hsa-miR-10a, hsa-miR-7a, and hsa-miR-302a) into the samples (Table 2). Accuracy was determined by comparing expected and observed concentration differences between serum sample 1 and 2 for all three miRNAs studied. TaqMan Advanced miRNA Assays more accurately quantified small differences in miRNA concentrations when compared to miRCURY LNA microRNA assays.

Target name

hsa-miR-10a

hsa-miR-7a

hsa-miR-302a

Expected

2.3

1

-1

TaqMan Advanced miRNA Assays

2.4

0.8

-1.2

miRCURY LNA micoRNA assays

2.9

0

-0.5

Table 2. Relative mean ΔCt values for synthetic miRNAs spiked into serum. Serum sample 2 was spiked with varying concentrations of three synthetic miRNAs, while serum sample 1 was spiked with fixed concentrations of the same miRNAs. The miRNAs were quantified by RT-qPCR and concentration differences expressed as relative expression (log2) values.